Increased expression of the human MDR1 gene and its product, P- glycoprotein, results in multidrug resistance in human tumor cells. To control the development of multidrug resistance, the mechanisms regulating the expression of this clinically important gene must be understood. The observation that two genes involved in mitogen-activated signal transduction, c-Ha-ras and v-raf, influence expression of the human and rodent MDR genes suggested that specific effectors and transducers of mitogen-dependent signalling might regulate MDR1 gene expression. Our preliminary experiments have identified several components of the Raf- mediated signal cascade which mediate transcriptional activation of the MDR1 promoter. Activity of the c-Raf-1 kinase is required for the effects of many serum mitogens and mediates the increased expression or activity of several transcription factors, including members of the ETS family. Our goal is to connect different members of the signal transduction pathway resulting in stimulation of MDR1 gene expression. The knowledge gained from these experiments will help to identify individual steps in the control of MDR1 gene expression which, if blocked, may prevent activation of MDR1 and thus block the development of multidrug resistance. Specific Aim 1 is to identify mitogen- and Raf-responsive elements through MDR1 promote mutagenesis. The results of these studies will provide insight into the specific mechanisms controlling the MDR1 response to mitogen stimulation. In Specific Aim 2, we will determine if Raf-1 kinase mediates all or part of the mitogenic response of the MDR1 gene. If Raf-1 kinase is the fundamental control point through which mitogen stimulation of MDR1 occurs, then tumor cells which have activated or amplified Raf might readily be selected for increased MDRI expression under appropriate growth or selective conditions. If other proto-oncogenes can independently activate MDR1 gene expression, then tumors bearing such activated or amplified genes might be primed for selection of increased MDR1 gene expression. Thus, identification of Raf-dependent and independent pathways is important. For these studies, a dominant-negative mutant of c-Raf will be used. Our preliminary results indicate that over two-thirds of the mitogen-activation of the MDR1 promoter is blocked by a dominant-negative Raf mutant, suggesting that c-Raf-1 plays a significant role in MDR1 activation. In Specific Aim 3 we will characterize the signals mediated through Raf-dependent transcription factors. Since c-Raf kinase activation occurs within minutes of mitogen treatment of cells, while the peak MDR1 response occurs 10 to 12 hours later, there must be intermediate factors which mediate the Raf-dependent MDR1 response. We will determine which, if any, of the Raf-dependent factors activates MDR1. Our preliminary results suggest that members of the ETS family of proto-oncogenes may activate MDR1 transcription. We will confirm and extend these studies by measuring the activity of MDR1 promoter mutants in response to expression of ETS genes. Further, we will identify and characterize cDNA clones of previously unidentified Raf-dependent activators that bind specifically to the MDR1 promoter.